Over the past decade, the use of immune deficient mice as recipients for human hematopoietic stem cell transplantation has dramatically improved stem cell research. Development of models to study the differentiation of human hematopoietic stem cells into most blood cell lineages has been accomplished and the systems have been optimized. However, robust systems to study development of one particular lineage of human blood cell, the T lymphocyte, have not yet been accomplished. The most reproducible in vitro system that allows human T cell development from purified stem cells; the fetal thymic organ culture, requires precise timed mating of murine breeders and then careful dissection of the resulting day 15 embryos. When successful, the technique produces very low numbers of human T cells. The best system currently available to study human T cell development in vivo, the scid/hu thy/liv mouse, requires procurement of human fetal tissues and accomplishment of survival surgery in mice, so is not available to many laboratories. Unfortunately, T cell development does not occur in the most popular murine xenograft recipient, the NOD/SCID mouse. In this mouse, transplanted human stem and progenitor cells are strongly and rapidly diverted toward B cell differentiation, and no human T cells are observed. Even mature human T cells are rapidly cleared following transplantation into NOD/SCID mice. In spite of the drawbacks to the existing systems, the scientific community constantly strives to improve these models because the ability to study production of large numbers of human T cells in mice would have an enormous impact on human health care. Delayed immune reconstitution is a major problem in stem cell transplantation, resulting in many deaths from post-transplant infection each year. A murine system to study post-stem cell transplant T cell reconstitution from purified human stem cells could allow dissection of the factors that could help transplant patients develop a functional immune system much more rapidly. The lack of a reproducible and effective T cell development assay that does not require surgical skill has been a major stumbling block for the stem cell field to date. We have discovered that co-transplantation of human mesenchymal stem cells engineered to express hepatocyte growth factor (HGF) with purified human stem cells causes massive human T lymphocyte development and expansion in NOD/SCID/B2M null mice. We hypothesize that human "thymopoiesis" is ongoing in all tissues where the HGF-secreting MSC co-localize with the stem cells. In support of this theory, the MSC/HGF/B2M mice have large numbers of double positive CD4+/CD8+ human cells in their spleen and liver, as well as thymus, but not bone marrow. These are the sites of human MSC homing in the mice. From our initial observations, T cell development appears to mimic normal thymopoiesis, with large numbers of double positive (CD4+/CD8+) human T cells produced in the organs, in addition to mature CD4+ and CD8+ cells in the circulation. The MSC/HGF/HSC cotransplanted mice form the most robust in vivo T cell development system that we have observed in the xenograft field to date. The current application proposes studies to refine this system, to understand it, and to develop it further to be a valuable resource for any investigator that wishes to study development of human T cells from hematopoietic stem cells in vivo.